Phazr-N3

Procedure (The following is our recommended protocol. This protocol is for reference only and should be modified according to your specific needs).

1. Stock Solution Preparation
Ethanol (EtOH).

2. Working Solution Preparation
2.1 Diluents:
2.1.1 Anhydrous solvent, water, 10 mM NaOH, PBS, culture medium.
2.2 Working concentrations:
2.2.1 200 nM in anhydrous solvent or water for fluorescence spectrometer spectral measurements.
2.2.2 200 nM in 10 mM NaOH for Aβ42 anisotropy measurements.
2.2.3 200 nM or 1 μM for Aβ42 plaque staining.
2.2.4 1 μM in PBS for plate reader aggregation measurements.
2.2.5 0.3 μM in DMEM containing 10% FBS and 1× penicillin/streptomycin for live HMC3/plaque microscopy.
2.2.6 200 nM or 300 nM in DMEM containing 10% FBS for confocal time-course.
2.3 Adjust working solution concentrations as needed; prepare fresh before use.

3. Staining Procedure
3.1 For Aβ42 aggregates/plaques[1]:
3.1.1 Sample types:
3.1.1.1 Synthetic amyloid β peptide (Aβ42) aggregates/plaques.
3.1.1.2 Pre-formed Aβ42 plaques; load plaque aliquots (50 μL) into separate channels of an imaging chamber and mount on plasma-cleaned glass slides.
3.1.2 Incubation conditions:
3.1.2.1 For Aβ42 aggregation monitoring: incubate 0.2 μM Phazr-N3 PBS stock solution with 20 μM Aβ42 at 37°C.
3.1.2.2 For pre-formed plaque staining: incubate plaque aliquots with 200 nM or 1 μM Phazr-N3 working solution at room temperature in the dark for 1 h with gentle agitation on an orbital shaker.

3.2 For Aβ40 aggregation assay:
3.2.1 Co-incubate 20 μM Aβ40 with 1 μM Phazr-N3 in PBS in sealed, black, non-binding 96-well plates at 37°C with continuous orbital shaking for 24 h.

4. Controls
4.1 Fluorescence standard: Cy5 (Φ = 0.27 in PBS) is used as the fluorescence standard for quantum yield calculations.

5. Detection and Analysis
5.1 Instrument types: fluorescence spectrometer, UV/visible spectrophotometer, plate reader, and confocal laser scanning microscope/confocal microscope.

5.2 Wavelength settings:
5.2.1 Solvent-dependent absorption/emission maxima: toluene 527/640 nm; EtOAc 529/657 nm; acetone 542/667 nm; CH3CN 546/680 nm; EtOH 571/692 nm; PBS 567/703 nm.
5.2.2 Quantum yield emission spectra: 600 nm excitation and 3-nm slit width.
5.2.3 Spectral shift and anisotropy: 580 nm excitation and 680 nm emission; anisotropy measurements use 580 nm excitation and 690 nm emission.
5.2.4 Cuvette spectra: 580 nm excitation.
5.2.5 Plate reader: 570 nm excitation and 680 nm or 695 nm emission.
5.2.6 Confocal microscope: 561 nm or 594 nm excitation and 600 to 720 nm emission; additionally 594 nm excitation and 630-710 nm emission; live-cell confocal microscopy uses 594 nm excitation and 600 to 724 nm emission.
5.2.7 Spectral imaging: collect emission from 628 to 704 nm at 3.2-nm or 3.9-nm intervals.
5.2.8 Signal-to-noise ratio measurements: 594 nm excitation and 603, 613, 623, 632, 642, 652, 661, 671, 681, 691, 700, 710, and 720 nm wavelengths.
5.2.9 Emission maximum shifts from 703 nm in aqueous buffer/PBS to 630 nm in toluene; another emission maximum at 640 nm in toluene is also reported.

5.3 Result analysis:
5.3.1 Phazr-N3 exhibits solvatochromism; fluorescence quantum yields are 0.03 in PBS, 0.11 in toluene, 0.16 in EtOAc, 0.27 in EtOH, 0.41 in CH3CN, and 0.62 in acetone. Emission λ_max values are 640 nm in toluene, 657 nm in EtOAc, 667 nm in acetone, 680 nm in CH3CN, 692 nm in EtOH, and 703 nm in PBS.
5.3.2 Fluorescence intensity changes: intensity increases 3-fold immediately upon mixing with Aβ42 and continues to increase over 12 h; plaque staining intensity is >4000-fold higher than background under no-wash conditions; intensity in plaque regions is higher than microglial or albumin background.
5.3.3 Fluorescence localization: localizes to synthetic Aβ42 plaques, including regions not stained by Thioflavin T; exhibits minimal nonspecific binding to live microglia or albumin.
5.3.4 Aβ42 plaque staining produces a high signal-to-noise ratio.